DESCRIPTION: (Investigator's abstract) Proteolysis is a fundamental mechanism in all living organisms that provides a sensitive way for titrating levels of active proteins essential for maintaining homeostasis and diverse critical cellular functions. The molecular components of the proteolytic machinery are incompletely understood. The central goal of this proposal is to achieve a better understanding of the intricacies of this machinery through the investigation of a novel lysosomal membrane protein named LAPTm5. In murine animals, LAPTm5 is specifically expressed in hematopoietic cells, and in human, it is preferentially expressed at high levels in hematopoietic cells. The protein was found to bind to ubiquinated proteins. There is accumulating evidence that ubiquination of proteins is an important signal for proteolysis in lysosomes. LAPTm5 has also been found to alter the level of IkB in lysosomes. IkBs are the key regulators of the activation of NF-kB, a major transcriptional factor responsible for regulating an extensive list of important genes including those involved in immune and inflammatory response, cell adhesion, stress response proliferation and differentiation. The process by which IkB is degraded and regulated is therefore an area of intensive research. The immediate goal of this proposal is to investigate the function of LAPTm5 from several approaches indicated by these findings. Aim 1 will determine if LAPTm5 can affect the NF-kB system by analyzing if it regulates IkB degradation in lysosomes, whether it can activate NF-kB and whether it has an influence on the responsiveness of the NF-kB system. Aim 2 will examine the role of LAPTm5 in hematopoietic cell development by using the model of in vitro differentiation of murine embryonal stem cells into hematopoietic cells. The effect of a constitutive expression of the gene and a disruption of the gene by homologous recombination will be analyzed. Aim 3 will use protein chemistry techniques, including 2-D gel analysis to determine the identity of the ubiquinated proteins that bind with LAPTm5. A direct in situ binding assay with purified lysosomes will also be used to identify binding proteins. New findings about this unusual gene will contribute to our knowledge about the molecular basis of the functions of proteolytic organelles and their relevance to hematopoietic cells. The proposal also may lead to new insight about the regulation of a key transcriptional factor that plays a significant role in many human diseases.